Production of a new protease

ABSTRACT

A new, peptide-forming type of alkaline protease which is characteristically different from any similar commercial product in substrate specificity, decomposition pattern, amino acid composition, elementary analysis and other physicochemical properties, and which has the greatest enzymatic activity between pH 11.0 and 11.7. The invention includes a method of preparing said enzyme by cultivating Bacillus subtilis var sakainensis (ATCC 21394), and by purifying the crude enzyme preparation obtained therefrom at a larger yield in comparison with other commercially available proteases, in crystal form, by a very simplified method.

United States Patent Inventor Sawao Murao Sakai, Japan Appl. No. 861,911 Filed Sept. 29, 1969 Patented Nov. 23, 1971 Assignee Sanraku Ocean Co., Ltd. Tokyo, Japan Priority Oct. 2, 1968 Japan 43/7 1084 PRODUCTION OF A NEW PROTEASE Primary Examiner-Alvin E. Tanenholtz Attorney-Holcombe, Wetherill & Brisebois ABSTRACT: A new, peptide-forming type of alkaline protease which is characteristically different from any similar commercial product in substrate specificity, decomposition pattern, amino acid composition, elementary analysis and other physicochemical properties, and which has the greatest enzymatic activity between pH 11.0 and [1.7. The invention 10 Claims, 1 Drawing Fig. U 8 Cl 195/62 includes a method of preparing said enzyme by cultivating "1.35/66 195/96, Bacillus bull-s varsakainensis (ATCC 21394), and by Punfy ing the crude enzyme preparation obtained therefrom at a Int. Cl C07g 7/028 larger yield in comparison with other commercially available Field of Search 195/62, 66 proteases, in crystal f by a very simplified method.

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PRODUCTION OF A NEW PROTEASE SUMMARY OF THE INVENTION This invention relates to a new alkaline protease and to the preparation thereof by cultivating a variety of spore-forming bacteria.

Proteases are commonly known as digestives or are utilized in the leather industry. But some proteases like chymotrypsin and. bromelin have recently proved extremely useful in other areas such as medical treatment and food and detergent additives. For example, they have gained public recognition for their efficacy when applied to medical treatment for wounded skin-lessening skin inflammation and removing discharges and deteriorated tissues from the inflamed region. It has been also known that they can be effectively employed as ingredients for meat tenderizers and for detergents for both dishwashing and laundry use. Accordingly, the demand for these prozeases has risen very rapidly.

Specifically, the growing popularization of dishwashers and the consumption of enzyme-containing laundry detergents will have a decisive influence on the market for proteases. Considering the above-mentioned possibilities and the industrial demand to be met, applicant has sought to find a new and superior protease, and this search resulted in the discovery of an especially potent strain selected from numerous micro-organisms isolated from nature, which would produce a new protease far superior to any known in its yield and purity, and then in identifying said strain by its mycological characteristics as a new variety of Bacillus sub'tilis.

The protease being produced from the above-named strain of microorganism was found to be a singular new kind, characteristically different from any known protease, similar in general properties, amino acid compositions, and substrate specificity, in particular. This protease was also found to be very easily crystallized in a few simple steps, as compared with other known proteases. This is a distinctive quality of this protease and is highly valued in its commercial applications.

lt is believed that this invention, relating to the new and superior protease and a method of its production by cultivating a specific strain of micro-organism should find application in a wide range of industries.

The mycological characteristics of the above-mentioned specific strain relating to this invention will be given below:

1. Morphological characteristics Vegetative cells: microscopic observation under a tempera ture of28C. to 32 C. for 18 to 24 hours Rods, 0.8 to 1.0 by 2.5 to 3.5 microns; in some cases long rods, 0.8 to 1.0 by 5.0 to 6.0 microns, or filaments. Single or occasionally in short chains (2-4). Not encapsulated, but coated with slime. Motile. Gram-positive. Not vacuolated if lightly stained. Sometimes, shadow forms. Growth on glucose nutrient agar is nearly same as on nutrient agar; but, cells contain a few small fat globules.

Spores, circular to cylindrical, 0.7 to 1.0 by 1.0 to 1.3 microns, thin-walled, central or paracentral. Usually formed in 48 hours. Sporangia frequently show bipolar staining; and very little, if any, swelling by spores.

11. Cultural characteristics:

Colonies on nutrient agar plates: (46 hours). Circular or irregular, undulated, convex or evenly raised, rough, dull, opalescent, offwhite.

Nutrient agar slants": (48 hours, 30 C.). Good growth at l to 2 days; spreading, glistening (center) to dull (margin), undulated, offwhite, opalescent, odorless, no pigment formed in inedium, smooth. After 7 to 24 days, offwhite with slight olive color, translucent, glistening.

Nutrient broth": 14 days, 30 C.). Slight turbidity and sediment, pellicle not formed at beginning but slightly formed at 7 or 8 days.

Nutrient gelatin slabs: (1 month, C.). Liquefaction crateriform in early stage to stratiform in 7 to 8 days. Nearly completely liquefied in 20 days.

F RAZIER gelatin agar plate: (3 days, 30 C.). Wide zone of hydrolysis.

Glucose nutrient agar slants": (48 hours, 30 C.). Same or slightly better growth than on nutrient agar. White, glistening, viscoid. Transparent at aging. No change in color of medium.

Soybean agar slants: (48 hours, 30C.). Same growth as on nutrient agar. White, smooth, glistening, spreading, lobate or arborescent.

Glucose asparagine agar slants: (48 hours, 30 C.). Moderate growth somewhat inferior to that on nutrient agar.

Glucose nitrate agar slants: (14 days, 30 C.). Scant growth after 2 weeks.

Potato plugs: (14 days, 30 C.). Good growth, creamy white, smooth, glistening, somewhat raised. After 14 days, still smooth and more yellowish.

NaCl broth: (14 days, 30 C.). Growth in 5 or 7 percent NaCl, but not decisive in 10 percent.

Litmus milk: (30 days, 30 C.). Slightly acid at 2 days, peptonized at 4 days; medium remains acidic.

Milk (agar streak) plates: (48 hours, 30 C.). Good growth,

white, wide zone of hydrolysis of the casein.

Salts (glucose agar streak): No growth, but moderate growth with biotin 10 to ug/l.

*DIFCO products 111. Physiological characteristics:

Temperature: Optimum between 28 and 40 C. Maximum,

50 C. No growth at 55 and 10 C.

Catalase-positive Starch hydrolyzed Acetylmethylcarbinol produced Gelatin hydrolyzed Casein hydrolyzed pH of glucose broth is 5.2 to 5.4 at 7 days, at 30 C.

Citrates utilized Nitrites produced from nitrates No gas produced in nitrate broth under anaerobic conditions.

Growth scant in glucose broth under anaerobic conditions;

pH is 6.1 at 14 days.

Lecithinase not produced.

Acid but no gas from arabinose, xylose, mannose glucose,

fructose, sucrose, maltose, starch, inulin, glycerin, mannitol, sorbitol. No acid or gas produced from lactose.

Source; isolated from soil and natto" (or fermented soybean) Referring to Bergeys Manual ofDeterminative Bacteriology, 7th edition (1967), the above-described strain of micro-organism was identified to be a type of Bacillus subrilis from general characteristics. However, it differs from Bacillus subtilis in the following:

Subsequently, the strain isolated according to this invention was defined to be a new variety of the above-referred strain and thus named Bacillus substilis var sakainensis.

A normal production of the proteolytic enzyme to which this invention relates can be attained by basically conventional microbial fermentation with the above-described strain; and submerged culture is the preferred method.

The medium to be used can be synthetic, semisynthetic or natural, but it should contain the nutrient sources which can be assimilated by the above-described strain. More specifically, a medium is composed of carbohydrates as a carbon source, e.g., commercially obtainable glucose, glycerin, sucrose,starch, dextrine, molasses and maltose-glucose syrup;

and organic and inorganic nitrogen materials as a nitrogen source, e.g., commercially available peptone, meat extract, corn steep liquor, cottonseed meal, fish meal, soybean flour, casein-hydrolyzing material, yeast extract, dried yeast, gluten meal, ammonium sulfate, ammonium nitrate, urea and so forth. Frequently, it also contains inorganic salts such as sodium chloride, potassium chloride, calcium carbonate and magnesium sulfate. In case excessive foaming occurs during the fermentation, a suitable defoaming agent can be applied.

The fermentation can be carried out at any temperature within the range in which said strain can grow and produce the desired enzyme. However, it can be most effectively carried out at between 25 and 37 C. The fermentation time in which a sufficient amount of the desired enzyme is accumulated in the medium, according to this invention ranges from 40 to 140 hours, varying with the modes of fermentation.

Naturally, the optima of these cultural conditions, i.e. temperature and time, are carefully selected for the fermentation according to the properties of the strain used. Taking a more concrete example, a seed culture is prepared by inoculating the strain, which has been precultured on a nutrient agar slant, in a nutrient broth (pH 7.2), and cultivating the broth by shaking it at 33 C. for 16 to 20 hours. Sometimes a percent soybean extractthe supernatant obtained by steaming grams of soybean meal in 100 ml. of distilled water, followed by extraction-is added to the nutrient broth, i.e. seed culture. Another nutrient broth used for the main culture medium contains 8 to percent carbohydrates such as dextrin, soluble starch and/or maltose-glucose syrup, in order to obtain a high yield of the protease. ln addition, the content of the nitrogen source, such as polypeptone or meat extract, which is usually 1 percent, may be brought up to 2 to 3 percent. This would subsequently cause an increase of the yield of said protease produced. The maximum amount of said protease, i.e., 5 to 8 mg./ml., can be produced from 100 ml. of the above-prepared culture medium which has been cultivated in a 500 ml. Sakaguchi flask on a shaker having a 7 cm. stroke at 120 rpm, at 33 C. for 4 to 5 days.

The enzyme accumulated in the medium when the cultivation is terminated is then collected in crystal form by various known methods. For example, the following methods are normally employed in appropriate combination: fractionation by solubilities, e.g., salting-out, precipitation by organic solvent or by isoelectric point; separation by adsorption; and still others such as dialysis, precipitation by certain metallic salts, removal of other proteins present in the medium by denaturing them, ion-exchange resin treatment, Sephadex treatment, electrophoresis, ultracentrifugation and so on.

The crystallization of enzymes by means of any known method is generally accomplished through very complicated processes. Even though proteases could probably be crystallized by relatively simple methods as compared with other enzymes, they still have to go through several processes. For example, one method would take the following steps: fractional precipitation by ammonium sulfate-dialysis-ion-exchange resin treatment-adsorption on cellulose-elutionprecipitation-dialysis-crystallization. Such complication naturally leads to uneconomical results, lowering the total yield of the enzyme and raising the production cost.

One of the features of this invention is to collect crude crystals of this proteolytic enzyme by a relatively simple method and still at a higher yield. A filtrate of the broth is fractionally-precipitated by ammonium sulfate while the pH of the broth is adjusted with an ammonium solution to approximately 8.5. The precipitates are collected either by centrifuging or by filtration, adjusted by adding an ammonia solution to pH 10.0, and dissolved in the minimum required amount of distilled water. The enzyme solution at high concentration is added with l 10 by volume of Palitzsch buffer solution (pH 8.2) and then is readjusted to between pH 8.2 and 9.0 by adding an acetic acid solution, and thereafter held to cool. The enzyme in crystals of long hexagonal plates or rectangular plates is separated therefrom. The activity of the enzyme thus obtained accounts for 30 to 50 percent of the total activity of the concentrated enzyme solution before crystallization. The remaining enzyme solution is further precipitated by a 50 percent saturation of ammonium sulfate and treated by the same method, i.e., dissolving in a small amount of water with its pH adjusted and cooling it. Repeating the above processes, the enzyme is further separated therefrom in crystalline forms, and the cumulative activity reaches to percent of the total activity of the original broth. Therefore, when suitable conditions are carefully chosen and applied, 5 to 6 grams of crude enzyme can be obtained from a liter of the broth. Such a simplified method of crystallization cannot be achieved with other known alkaline proteolytic enzymes produced from Bacillus subtilis. The crude enzyme thus obtained is further purified by washing, dissolving, pH adjustment by adding a borate buffer, and cooling. The yield of the pure final product is as high as 60 to 70 percent. Other than the above-described methods, said enzyme can be crystallized and separated out of the filtrate by simply adjusting the pH of the filtrate. But in that case, the yield is merely 2 to 3 percent of the total activity.

In addition to the above-described procedures, the use of a Sephadex column simplifies the process even further. Such solvents as 60-70 percent acetone, ethanol and methanol may also be used in the precipitation method. The crystals ob tained by using these processes are of varied purity, ranging from 10 to percent. The purity of the product is not restricted by this invention.

The general enzymatic characteristics as well as the physicochemical properties of the enzyme produced according to this invention will be described in the following pages, which show that said enzyme is of a new type and quite different from other known types.

The activity of the protease according to this invention is estimated by the Casein-Folin method. For example, 1.0 ml. of the sample protease-diluted with buffer solution and adjusted to bring the ultimate activity (measured by optical absorption at 660 mp.) within 0.1 to 0.5-is treated with 5.0 ml. of 1.2 percent casein solution, and the mixture is allowed to stand at 37 C. for 10 minutes. Then with 5.0 ml. of reagent B (a mixture containing 0.1 l Mols of CCl COOH, 0.22 Mols of CH COONa and 0.33 Mols of CH COOH) or reagent A (0.44 Mols of CCI COOH) added, it is held for an additional 30 minutes at 37 C. and thereafter filtered. The enzymatic activity of the filtrate is estimated by optical absorption at 275 mu. This process is called a simplified" method in describing this invention. One ml. of the above filtrate is then added with 5 ml. of 0.44 Mols of NaCO solution and 1 ml. Folin reagent (X5 dilution), and the mixture is allowed to stand for 20 minutes at 37 C. Then the activity is estimated by optical absorption at 660 mp. This is the so-called Casein-Folin method. For comparing enzymatic activities, the values expressed by the optical absorbancy at 660 mp. are directly applied, but the activities are usually expressed by the numerals converted to a value of 1 ml. of the original sample or of 1 mg. of the solid sample.

The enzymes used for comparison in this invention are: a commercial product produced from Bacillus subtilis, and sold under the trade name Nagarse, which has been recrystallized three times (and is hereinafter referred to as enzyme N), and an alkaline protease produced from Bacillus subtilis var amylosacclzariricus, which has been recrystallized three times (hereinafter referred to as enzyme A).

( 1 Mode of action The proteolytic activity of the enzyme according to this invention was assayed by the Casein-Folin method at various pH values ranging from 6 to 13. As shown in table 1 below, the activity of said enzyme on casein is particularly high between pH 10 and 12, and its optimum is at pH 11.0 to 11.7. The above fact indicates that said enzyme is an alkaline proteolytic enzyme like the other enzymes, N and A, used for comparison. However, the optimum pH for the activity of said enzyme is even more alkaline than those for the others which are pH Table l 5 relationships of pH to proteolytic activity Note: The enzyme solution is prepared by recrystallizing the crystals having the highest activity according to this invention five times (this procedure is used for all the experiments hereinafter described, unless otherwise noted), and dissolving these crystals in water, 0.07 mg./ml. 0.5 ml. of each of the above solutions is withdrawn and 2.5 ml. of 1.2 percent casein solution is added thereto at the appropriate pH (i.e., pH 5.8-8.2 adjusted with KH PO Borax, pH 9.2-10.4 with Borax-Na Co and pH l l.0l2.7 with glycine-NaCl-NaOH. Then, with 2 ml. of reagent A, the activities are estimated by Casein-Folin method as described hereinabove.

In addition to the above, said enzyme demonstrates a certain hydrolysis pattern with casein used as substrate, which is different from those of other known alkaline proteolytic enzymes. The comparison is made in table 2.

Table 2 Hydrolysis Patterns of Three Enzymes using Casein as Note: The simplified method as hereinbefore described is employed. A and B in the above table express the activities determined by reagents A and B respectively. And, specifically, A expresses the amount of amino acid produced as result of hydrolysis; and B, the total amount of hydrolysis, i.e., those of peptide and amino acid. Therefore, (BA)/B indicates the ratio of peptides produced against the total amount of hydrolysis. (Hagihara: ibid., 305) It is known that proteolytic enzyme produced from Bacillus subtilis has a low substrate specificity, which indicates it is an amino-acid-producing type. This is shown by the enzyme N as in table 2. But the new enzyme which is also proteolytic produced from a strain of Bacillus subtilis has a higher substrate specificity, even closer to that of trypsin, thus indicating it is a peptide-forming type. Furthermore, these differences (substrate specificity and proteolytic pattern) between the new enzyme and the commercially available enzyme N become more obvious when the hydrolyzed substance of casein after hydrolysis by those enzymes are respectively fractionated on a Sephadex G--25 column. As in table 3, the substance hydrolyzed by enzyme N shows a higher peak for the 75 low-molecular, i.e., amino acid fraction, while that hydrolyzed by the new enzyme shows a peak only for the macromolecule, i.e., peptide fraction.

Table 3 New Enzyme Fraction Enzyme N Fraction A (high peptide) 76% 53% Fraction B (low peptide amino acid) 24 47 From the above, it can be concluded that the new enzyme has the optimum pH further toward the alkali side than any of the known alkaline proteases and is peptide-forming having the required substrate specificity, while the others are aminoacid-forming having no or low substrate specificity. And these properties single this new enzyme out of other alkaline proteases, as an entirely new enzyme.

(2) Substrate Specificity Using various proteins, the substrate specificity of the enzyme utilized in this invention was tested in comparison with the enzymes A and N, and the results are shown in table 4 below.

Table 4 Study on Substrate Specificity Relative Reaction Initial Rate Substrate new enzyme A N Milk casein Hill IOU Egg white albumin 43 6 J3 Gelatin I66 I55 I48 Soybean protein 54 59 4t) Gluten I09 97 R7 Haemoglobin I02 1 I2 I04 Note: 20 ml. of the mixture. in which various proteins 100 mg.) and enzymes (2 mg. each) are dissolved in M boric acid-sodium hydroxide buffer, is allowed to stand for 30 hours at 37 C. and thereafter assayed by the ninhydrin method.

As the above table shows, a large degree of disparity among the three enzymes is seen in the initial rate of the reaction on egg white albumin; that of the new enzyme is distinctively larger than that of A, and somewhat larger than that of N.

According to M. Ottesen et al. (Compt. Rend. Trav. Lab. Carlsberg, 34, 199: 1964), the alkaline proteases produced from B. subtilis can be generally divided into two major types in substrate specificity: (l subtilopeptidase A which demonstrates specificity towards such substrates as those containing L-leucine, and (2) subtilopeptidase B whose specificity is directed to the substrates containing L-tyrosine. It is accepted that enzyme N belongs to this latter type.

The substrate specificity of the new enzyme was further tested in comparison with enzyme N (subtilopeptidase B) by the pH-Stadt method using the following synthetic substrates: acetyl-X-ethylester (X represents glycine, L-valine, L-norvaline, L-leucine, L-tyrosine, L-phenylalanine and L-lysine) and benzyl-L-arginine-ethylester. The new enzyme exhibited the maximum activity on acetyl-L-leucine-ethylester and considerable activity on acetyl-L-tyrosine-ethylester and acetyl-L- phenylalanine-ethylester, respectively. On the other hand, enzyme N reacted most on acetyl-L-tyrosine-ethylester but very little on acetyl-L-leucine-ethylester. The above results further distinguish the new enzyme from enzyme N and put it into the subtilopeptidase A type.

In conclusion, the hydrolytic activities of three enzymes on various synthetic peptides and amino-acid ester chains used as substrates are summarized systematically as in table 5 below:

Table 5 Study on Substrate Specificity New Enzyme Enzyme No. Substrate Enzyme A N (Amino Acid esteis) lL-phenylalanyl-ethyl 2 L-tyrosyl-ethyl 3 N-acetyl-L-tyrosyl-ethyl (Dipeptides) 4 Glycyl-glycine 5 GlycyLDL-alanine 6 Glycyl-DL-valine 7 Glycyl-L-leucine 8 Glycyl-L-proline 9 Glycyl-L-tyrosine l Glycyl-DL-phenylalanine l I DL-alanyl-glycine l2 DL-alanyl-DL-valine l3 L-leucyl-L-tyrosinc l4 DL-leucyl-DL-leucine l Histidyl-histidine l6 L-leucyl-bphenylalanine l7 N-CBZ'L-vulyl-L-alanine 18 N-CBZ-L-valyl-L-proline l9 N-CBZ-L-valyl-L-leucine 20 N-CBZ-L-glycyl-L-tyrosinc 2| N-CBZglycyl-L-phenylalanine 22 N-CBZ-glycyl-L-phenylalanineamide 23 N-CBZ-leucyl-L-leucineamide 24 N-CBZ-tyrosyl-glycineamide (Tripeptides) 25 Glycyl-glycyl-glycinc 26 Glycyl-L-prolyl-L-alanine 27 L-leucyl-glycyl-glycine 28 DL-ulunyl-glycyl-glycinc 29 DL-leucyl-glycyl-DL-phenyl alanine 30 Glututhione (glutumylcystinylglycinc 31 N-CBZ-Lwalyl-glycylglycine-benzylester (Tctrapeptide) 32 Glycyl-glycyl-glycylglycine Note: A mixture of a substrate (2.0 mg.) and borate buffer (0.4 ml., pH 8.5, containing 100 ug. of enzyme) was left to react for 20 hours at 37 C., and a suitable amount of the reaction mixture was assayed by either a thin-layer chromatography with silicagel or by paper chromatography to identify amino acids and derivatives thereof obtained as results of the enzymatic reaction.

In table 5, the differences in substrate specificity among three enzymes are seen with respect to numbers, 2, 3, 24 and 27. That is, the new enzyme differs from N in activity rate in reacting towards the carboxyl side of L-tyrosine (Nos. 2, 3 and 24); and the new enzyme reacts on the carboxyl-radical side of L-leucine to decompose the peptide link while the other two do not (No. 27).

(3) Effect of pH on the enzymatic activity and stability Using casein as the substrate, the effect of pH on the enzymatic activity was tested with the enzyme according to this invention, and the results are shown in table 6.

Table 6 Effect of pH on the Enzymatic Activity Note: The following butters were employed for the adjustment of various pH values: M/50 sodium veronal, M/50 hydrochloric acid (pH 6.5-8.6) M/2O sodium borate, M/20 sodium carbonate (pH 8.9-l0.8) M/ 10 sodium hydroxide, M/lO disodium phosphate (pH 9.9-l2.5) Other conditions are same as described in the note for table 1.

Summarizing the above, the optimum pH for the activity of said enzyme at 37 C. is between 10.8 and 12.0, and the activity is maintained at 80 percent at pH 10.0 and nearly halved at pH 6 to 9 and at 12.5. The activity of said enzyme (per mg. of protein) at various pH values is further compared with the other enzymes, A and N, as in table 7 below wherein the relative activities of said enzyme and A are compared with the activity of N expressed as 100. Again, as is clear in the table below, the specific activity of said new enzyme (per mg.) differs from those of the others.

Table 7 Specific Activities of the Enzymes at Various pH values pH values Enzyme 7.4 8.5 10.5 11.5 Opt.

N 100 100 100 100 A 87 88 89 I40 90 New 62 65 73 80 Lastly, the efiect of pH on the stability of the new enzyme is shown in table 8, below. The residual activity of the reaction mixture is estimated after the enzyme solution has been incubated for 20 hours at 30 C. for reaction with various pH buffer solutions.

Table 8 Effect of pH on the Enzymatic Stability Residual Activity pH (Index No.)

4.0 negligible 5.0 negligible 6.0 negligible 45 8.0 98 9.0 98 9.5 98 10.5 98 10.8 98 l 1.0 75 l 1.5 35 l 1.8

negligible The te'mperature-activity relationship of the enzyme according to this invention was examined, and the result is shown in table 9, below:

mined at pH 8.5 after the times of storage, with toluene at the specific temperatures and specific periods of time given in the following table:

Table 9 TABLE B.COMPARATIVE STUDY ON STABILITY AGAINST STORAGE Temperature and Enzymatic Activity Resid' activity percent after Enzyme 10 days, 5 C. 10 hr., 30 C. hr., 30 C. Enzymatic Addition 10 Temperature Activity of Calcium g enzyme 23 gg 8 C. (index No.) lon (M/ZSO Cu") 9O 50 0 40 22 45 42 (6) The effects of pH and temperature on enzymatic action :2 g; :2 As discussed above, the new enzyme according to this in- 60 100 I vention remains perfectly stable between pH 8 and 10.5, at 65 95 :20 C. But at pH 7 or 1 1, its stability is somewhat lost, and 70 20 149 20 further at pH 6 or 12, it is completely lost. In relation to tem- Zg Negligible if: perature, said enzyme in solution at pH 8.2 shows stability for 90 Negligible 25 a short time at 40 C. or below. But it becomes somewhat unstable between 45 and 55 C. and extremely so between 60 and 65 C.; and finally it is inactivated at 70 C. As for a longer Note: pH 10.5 adjusted with borate buffer solution; reaction 25 Storage Stability Said enzyme remains highly Stable at a 10W time minutes temperature, e.g., 5 C. for 10 days; and is still 90 percent ac- Summarizing the above, h new enzyme reacts between tive after 20 hours, at 30 C. The other enzymes tested in comroom temperature and 70 C.; but the optimum temperature Parison under the conditions for 20 hours) gave for the enzymatic reaction is 55 to 65 C., and the maximum totally negative results- 60 C. In the presence of the calcium ion (4 l0 M), how- 30 (7) Inhibitors and their effects ever, the stability of said new enzyme increases to the First, the effects of various buffer solutions on the activity of imum of I40 percent at 70 C. 0n the other hand, the reaction the enzyme according to this invention were examinei and of enzyme A reaches its maximum at 55 C. (pH 10.5) and, in the results are Show" in table 1 it below the presence of the calcium ion (4Xl0 M), at 62 C. with only a 70 percent increase. (Fukumoto, Agricultural and Table l l Biological Chemistry, 30, I261: l966) The table below shows the stability of said new enzyme against temperature:

Effects of Bufi'ers on Enzymatic Activity Table 10A 0 Buffers pH 7.4 pH91) Temperature Stability Wsmimcl M/20 Nu,ta,o,-M/s 14,110,. Temperature Residual Actlvlty Mlzo MIC 93 83 45 M/l5 xH,Po,-M/1s mmpo, 88-91 66 7l M/lO Veronul-M/IO HCI 80-82 77-85 35 [00 MIN) Nu,co ,-M/10 H m... KCl 90 74 40 MIN) NH,OH-M/l0 NH,cl. 100 as so as 55 12 60 l 5 The above results show that the activity of said enzyme IS in- 65 5 hibited by the following buffers: Siirensen's KH PO -Na, 70 lz z HPO and NH OH-NH Cl buffer solution, at pH 9.0. Then,

again, the activity of said enzyme was tested with various inhibitors under the following conditions: pH 7.4 and 9.0, at 37 Note: The enzyme is dissolved in Palitzsch buffer solution C., for 30 minutes. Table 12 below gives the results as follows:

(pH 8.5) and left to react for 15 minutes.

Furthermore, the enzyme according to this invention ex- TABLE 12.EFFECTS 0F INHIBITORS ON ENZYMATIC hibited a great deal higher stability than the other known ACTIVITY IN COMPARISON WITH ENZYME A proteolytic enzymes produced from Bacillus subtilis, after a Newemyme longer storage period. As shown in table 108, there is no 00mm EnZYmeA change at all in the activity after the enzyme is left to react in Inhibitor nation (pH M) (pH (DH Palitzsch borate buffer (pH 8.2) for 10 days at 5 C., while the 100 100 100 other enzymes, N and A, lose 48 percent and I0 percent of HgC]; 1 10-aM their activities respectively. Again, when allowed to react for ggg ifigjgx 20 hours at 30 C., the enzymes, N and A, are totally inac- EDTAII: 2X10 M tivated, while the new enzyme loses only 10 percent of its total gig-:1: activity. As for the enzymes, N and A, used for this experi- PCMB 2x10 M ment, the commercially available sample crystals which cong f fj gfigifi tain some inactive material (approximately 30-40 percent), as Ascorbic acid 2X10- M shown in the FIGURE, are subjected to gel filtration prior to W the stability test against storage. [The portion of the active Note: DFP-di-isopropylfluorophosphate peaks of the respective enzymes are added with lO-time EDTA-4-acetic acid volume dilution of Palitzsch borate buffer (pH 8.2) to adjust MIA-monoiodoacetic acid SLS--Sodium lauryl sulfate the protein content] and their residual activities are deter- PCMB-parachloromercury benzoic acid BKClchloride The above inhibitors were respectively added to the reaction mixture containing 20mM Tris-HCl buffer (pH 7.4) and borate buffer solution (pH 9.0), and the mixture was allowed to stand at 37 C. for 30 hours.

From the above results, it is clear that the enzyme according to this invention, which is completely inhibited by DFP, belongs to the serine enzyme group having serine at the center of their activity. And secondly, the activity of the new enzyme is less inhibited by metalic salts than enzyme A.

(8) Purification Reference is made to the previous description and to the examples hereinafter set forth for the details in this point. As described therein, the simplified and economical method is employed, by which 4 to g. of crude enzyme is collected from a liter of the filtrate of cultured broth by fractional precipitation with ammonium sulfate, according to this invention. Then after recrystallization of the above crude crystals three times, 3 to 3.5 g. of the pure crystal enzyme is obtained. The accumulation of the enzyme in this invention is as high as 6 to 8 mg. per ml. of the medium, the crystallization of which enzyme can be simply accomplished by adjusting the pH of the medium. Considering this feature only, therefore, the advantage of this invention is clearly seen in its high yield of the enzyme and in the simple process of purificiation by crystallization.

(9) Physicochemical characteristics TABLE 13 Table l 4 Mobility in Electrophoresis (unit cmP/V sec.)

Item New enzyme Enzyme A pH Mobility p.

8.55 7.3lXl0'AH6 9.0 4.45xl0'AH6 9.5 3.28Xl0AH6 10.0 0

l5 12) Elementary analysis Reference is made to item 9. l3) Composition of amino acids The amino acid composition of the enzyme according to this invention is compared with those of other enzymes as in table 15, below:

Summarizing the above, the amino acid composition of the enzyme according to this invention is distinguished from that of enzyme A BY its percentage of such amino acids as lysine,

histidine, aspartic acid, serine, glycine, alanine, valine, leucine, and tyrosine; and from enzyme N and subtilopeptidase B by its percentage of lysine, histidine, arginine, threonine, isoleucine, serine, and tyrosine. It is differentiated from subtilopeptidase A which resembles said new enzyme in its subtilopeptidase Enzyme N A B Experimenta 11. 812. 2

Calculated. 12. 0 Sedimentation (10- sec.) 2. 94 Partial specific volu Experimental. 0.76

C ulculated 0. 73 ISO-electric point p1{) 10. 0-10.15 7. 5-8. 0 Molecular weight 29.0)(10 -30.0X10 22. 7X10 Friction ratio, f/fo 1. 17-1. 18 1. 04 Specific activity (unit/mg. protein). 2.4)(10 2.3)(10 Terminal amino acid Alanine Alanine Elementary analysis, percent:

N 14-15 15. 00 Ash 0.3-0. 7 1.

27. 7x10 30. 0X10 1. 18 1. 14 2.2)(10 Alanine Note: The source for the data on the enzymes A, N and Subtilopeptidase A and B is: Fukumoto, Agriculture and Biological Chemistry, 31,330: 1967. The sample enzymes used for the experiments are crystals which have been recrystallized three times or lyophilized samples thereof. For the ultracentrifuge, electrophoresis, and intrinsic viscosity tests, the samples were dialyzed overnight in a cool place against appropriate buffer solutions.

(10) Molecular weight The molecular weight of the enzyme according to this invention was calculated as below, following the formula by Sheraga and Mandelkern:

Sedimentation coefficient S=3.23 Intrinsic viscosity [u]=0.033 dl./g. Viscosity ofsolvent v=0.9032 Partial specific volume V=0.73 cm./g. Axial ratio =4 B=2.20 X10 Mol. wt. 29,000-30,000 The molecular weight of the new enzyme obtained by the above process can be compared with those of the other enzymes, A and N, in table 13, which are 22,700 and 27,700 respectively. l l Mobility in electrophoresis Table 14 shows the mobility in electrophoresis of the enzyme according to this invention at pH 8.5 to 10.0

TABLE 16.AMINO ACID COMPOSITION OF ENZYMES IN COMPARISON subtilopeptidase New Enzyme Enzyme Ammo acid enzyme A N A 13 Lysine ll 6 11 E1 11 Histidine 6 5 6 5 6 Arginine 4 3 2 4 2 28 20 28 31 20 18 14 14 20 13 41-44 37 38 37 38 13 10 14 J 15 16 12 16 12 15 38 25 34 3.) 34 36 27 38 43 38 0 0 0 0 0 30 2!) 30 35 31 5 3 5 (i 5 17 12 13 11 13 16 12 15 17 15 12 0 10 13 10 3 2 3 4 3 Tryptophene 4-5 3 4 3 1 Molecular wt. 27,700 30,000 28,000

behavior with synthetic substrates by the residual quantities of asparatic acid, serine, proline, glutamic acid, alanine, valine, trylophan.

From the various aspects described above, said enzyme strictly speaking can be distinguished from all other enzymes so far known and used for comparison, in terms of its enzy- Five ml. of soybean extract prepared in accordance with the procedures hereinbefore described is added to 100 ml. of nutrient broth containing 1.0 percent polypeptone, 1 percent meat extract and 0.3 percent NaCl. This is used as a seed culture medium, in which a loop of the culture of this strain relating to this invention precultured on a nutrient agar slant is inoculated. After an overnight incubation on a shaker at 33 C., it is ready to be used as a seed culture.

The main culture medium is prepared by adding 10 percent soluble starch to the above-mentioned nutrient broth, and, after sterilization, it is poured into 260 Sakaguchi flasks having a capacity of 500 ml. Two ml. of the above-prepared seed culture is inoculated into each flask of the main culture medium and incubated on a shaker at 33 C.

During the cultivation, the pH of the medium, the growth of the cells, and the enzymatic activity were measured in relation to the period of cultivation as shown in Table 16.

TABLE 16 Cultivation period (days) pH 6.8 6.4 6.4 6.3 6.2 Growth of the cell (absorbancy at 610 m 13. 2 17.3 21.0 21.0 20. 4 Enzymatic activity (absorbancy at 660 m 4. 6 13.3 19. 1 26. 2 25.3

The enzymatic activity was determined with borate buffer solution (pH 8.2) as hereinbefore described in this specificat|on.

When the cultivation is terminated. the culture broth is centrifuged to remove the cells and thereafter filtered. Ammonium sulfate is added to the 5.3 l. of the filtrate to make a 50 percent saturated solution while the filtrate is being adjusted to pH 8.5 to 9.5 with a 10 percent aqueous ammonia, thereby separating the enzyme protein. After the above has been held overnight in refrigerator, the precipitate is collected by centrifuge and dissolved in 500 ml. of distilled water while the pH is being adjusted with 10 percent aqueous ammonia to around 9.0 to 10.0. Then, the insoluble constituents are removed by centrifuge, and the supernatant is adjusted to pH 8.2 and held overnight in refrigerator, thereby separating the enzyme protein in crystal form.

The crystals obtained were collected by centrifuge, suspended in a l/lO Palitzsch borate buffer, again and repeatedly centrifuged, and finally rinsed. Thus, 14.6 grams of crude enzyme crystals were obtained. This yield is 58 percent of the total activity of the filtrate.

The mother liquid, together with the water with which crude crystals have been washed, is 50 percent saturated with ammonium sulfate, and the precipitate formed is collected by centrifuge. Following the procedures above described, 9.5 grams of crude crystals of the enzyme according to this invention was recovered from the mother liquid. Together with the previous yield, it amounted to 92 percent of the total activity.

EXAMPLE 2 To 2.5 liters ofthe filtrate as obtained in example 1 is added 3.5 liters of acetone; the precipitate obtained is dissolved in 500 ml. of distilled water (pH 9.0) and further treated with ammonium sulfate to bring into 50 percent saturation. The precipitate is collected by centrifuge, and is crystallized according to the procedures as described in example 1. The yield of the crude enzyme before the mother liquid is recovered was 42 percent of the total activity.

EXAMPLE 3 Twenty four grams of the crude crystal enzyme as obtained in example 1 was dissolved in a small amount of distilled water, while the pH was maintained between 9.0 and 10.0 with aqueous ammonia. Again adjusting the pH at 8.2 with acetic acid, it was recrystallized by holding it in a cool place. The crystals thus obtained were collected by centrifuge and rinsed repeatedly. As a result, 13.6 grams of the pure crystals were obtained.

EXAMPLE 4 One hundred ml. of culture medium composed of 0.1 percent, K HPO 0.3 percent NaCl, 0.05 percent MgSO,-7H,O, 2.0 percent fishmeal, and 8.0 percent maltose-glucose syrup at a pH of 7.2 was introduced into a Sakaguchi flask and sterilized. Two ml. of the seed culture prepared as in example 1 was inoculated and cultivated for 5 days under the same cultural conditions as given in example 1. At the end of cultivation, 5.6 mg. per ml. of enzyme was accumulated in the medium.

EXAMPLE 5 Cultivation under the same conditions as in example 1 was carried out in a medium comprising 2.0 percent polypeptone, 2.0 percent meat extract, 20 percent maltose-glucose syrup and 0.3 percent NaCl. After 5 days, 8.2 mg. per ml. of enzyme had accumulated in the medium.

EXAMPLE 6 Five hundred ml. of the culture broth obtained in example 5 was adjusted to pH 8.5 by adding 50 ml. of Palitzsch borate buffer solution (pH 8.2) and held overnight in a cool place to separate the enzyme crystals. The crystals obtained were centrifuged, rinsed and dried. As a result, mg. of crude crystals were obtained, which accounted for 2.8 percent of the total activity.

Explanation of some symbols and units of physical constants used in this specification: l. Sedimentation coefficient, s20w Order of sedimentation coefficient is l0"sec., and not l0 '"/sec.

s20w means a value determined in terms of 20 (3., using water as a solvent.

2. Friction ratio, f/fo jfrictional coefficient; f/fi)"frictional ratio Frictional force of a particle is proportioned to sedimentation rate, and its proportion constant is represented by f. of is frictional coefficient of a spherical particle of the same value as the above-referred particle and having no hydration.

The term, f/fo, is often used in determining a frictional ratio and gives specific values to various substances.

3. Intrinsic viscosity, [1;]

Viscosity is usually expressed by g./sec.cm.

However, intrinsic viscosity is entirely of another dimension, and the use of the word, viscosity, may not be appropriate. Therefore, [1;] is expressed mostly by cmslg. and sometimes by dl./g., in which d1. is of course deciliter.

4. Partial specific volume, [V 1 It is defined as a voluminal increase of the liquid when 1 gram of a certain substance is dissolved in an ample amount ofliquid, and it is expressed by cm./g.

What is claimed is:

1. A new alkaline protease which is characterized by it capability of being crystallized by simple process and which has the following properties:

a. Substrate specificity such as affinity for the carboxyl-radical side of L-leucyl of the peptide and ester bonds being stronger than affinities of other enzymes;

b. Active pH between 6.0 and 13.0, optimum pH from 10.5 to 11.0, stability maintained between pH 8.0 and 10.5,

and inactivation at pH 6.0 or below and pH 12.0 or above (at 30 C.

c, Active temperature between room temperature and 70 C., optimum temperature between 55 and 65 C., stability maintained at between 5 and 30 C., and inactivation at 70 C. or above (pH 8.2);

d. Serine protease type having serine at its center of activity,

which is inhibited by di-isopropyl fluorophosphate;

e. The following physical and chemical properties:

Absorbancy (Elcm.) R80 my. Sedimentation coefl'lcient (r20w) Intrinsic viscosity (1 Partial specific volume Isa-electric point Molecular weight 29.0X l-30.0Xl0 Friction ratio (f/fu) l.l7-l l 8 Specific activity 2.4x I0 f. Elementary analysis: C, 47-48percent; H, 6.5-7.2 percent; N, 14-15 percent; and Ash, 0.3-0.7 percent; and g. The following amino acids composition:

Lysine 9 Histidine 6 Arginine 4 Aspartic acid 28 Threonine l8 Serine 4]44 Proline l3 Glutamic acid [6 Glycine 38 Alanine 36 Half-cystine 0 Valine 30 Methionine lsoleucine l7 Leucine l6 Tyrosine l2 Phenylalanine 3 Tryptophan 4-5 Molecular weight 30,000

2. A process for the production of a new alkaline protease as claimed in claim 1, which comprises steps of inoculating Bacillus subtilis var sakainensis (ATCC 2l394) in a medium containing at least one carbon source selected from the group consisting of glucose, glycerine, cane sugar, starch, dextrin, molasses, and glucose-maltose syrup, at least one nitrogen source selected from the group consisting of peptone, meat extract, CSL, cotton seed meal, ammonium phosphate, urea, and ammonium nitrate, and inorganic salts; of cultivating the said strain of micro-organism under aerobic conditions at pH 6.0 to 8.0 and at a temperature between 25 and 37 C.; accumulating the new alkaline protease in the medium; and purifying said protease by combining more than one process selected from the group consisting of salting-out, precipitation with solvent; precipitation by l.E.P., absorption, dialysis, ionexchange resin treatment, and Sephadex treatment.

3. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing 1 percent polypeptone and 1 percent meat extract as a nitrogen source 10 percent soluble starch as a carbon source, and 0.3 percent sodium chloride as an inorganic salt is employed.

4. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing fishmeal as a nitrogen source, glucose-maltose syrup as a carbon source, and 0.1 percent K HPO 0.3 percent sodium chloride and 0.05 percent magnesium sulfate as inorganic salts is employed.

5. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing 2.0 percent polypeptone and 20 percent meat extract as a nitrogen source, 20 percent glucose-maltose syrup as a carbon source, and 0.3 percent sodium chloride as an inorganic salt is employed.

. A process for the production of a new alkaline protease,

according to claim 2, in which a purification comprises the steps of fractionally precipitating the filtrate of the culture broth with ammonium sulfate at pH 8.5, collecting the precipitate by centrifugation or by filtration, adjusting thepH to 10.0, dissolving said precipitate in the minimum required 7. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of precipitating the filtrate of the culture broth by adding acetone, dissolving the precipitate in the minimum required amount of distilled water, fractionally precipitating with ammonium sulfate, collecting the precipitate by centrifugation or filtration, dissolving said precipitate in the minimum required amount of distilled water at pH 9.0, readjusting the solution to between pH 8.2 and 8.5 with Paliztsch borate buffer (pH 8.2) or acetic acid solution, and finally separating the enzyme produced in crystal form.

8. A process for the production of a new alkaline protease, according to claim 2, which comprises steps of adjusting the pH of the filtrate of the culture broth to 8.2 with Paliztsch borate buffer or acetic acid solution, and separating therefrom the enzyme in crystal forms.

9. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of fractionally precipitating the filtrate of the culture broth with ammonium sulfate, collecting the precipitate by centrifugation or filtration, adjusting the pH to 9.0, dissolving it in the minimum required amount of distilled water, and purifying on a Sephadex column.

10. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of precipitation the filtrate of the culture broth with a solvent selected from the group consisting of acetone, methanol and ethanol, adjusting the precipitate to pH 9.0, dissolving it in distilled water, again precipitating it with the above solvent, and separating the pure enzyme crystals. 

2. A process for the production of a new alkaline protease as claimed in claim 1, which comprises steps of inoculating Bacillus subtilis var sakainensis (ATCC 21394) in a medium containing at least one carbon source selected from the group consisting of glucose, glycerine, cane sugar, starch, dextrin, molasses, and glucose-maltose syrup, at least one nitrogen source selected from the group consisting of peptone, meat extract, CSL, cotton seed meal, ammonium phosphate, urea, and ammonium nitrate, and inorganic salts; of cultivating the said strain of micro-organism under aerobic conditions at pH 6.0 to 8.0 and at a temperature between 25* and 37* C.; accumulating the new alkaline protease in the medium; and purifying said protease by combining more than one process selected from the group consisting of salting-out, precipitation with solvent; precipitation by I.E.P., absorption, dialysis, ion-exchange resin treatment, and Sephadex treatment.
 3. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing 1 percent polypeptone and 1 percent meat extract as a nitrogen source 10 percent soluble starch as a carbon source, and 0.3 percent sodium chloride as an inorganic salt is employed.
 4. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing fishmeal as a nitrogen source, glucose-maltose syrup as a carbon source, and 0.1 percent K2HPO4, 0.3 percent sodium chloride and 0.05 percent magnesium sulfate as inorganic salts is employed.
 5. A process for the production of a new alkaline protease, according to claim 2, in which a medium containing 2.0 percent polypeptone and 2.0 percent meat extract as a nitrogen source, 20 percent glucose-maltose syrup as a carbon source, and 0.3 percent sodium chloride as an inorganic salt is employed.
 6. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of fractionally precipitating the filtrate of the culture broth with ammonium sulfate at pH 8.5, collecting the precipitate by centrifugation or by filtration, adjusting the pH to 10.0, dissolving said precipitate in the minimum required amount of distilled water, adding to the enzyme solution 1/10 by volume of Palitzsch borate buffer solution (pH 8.2), and readjusting said enzyme solution to between pH 8.2 and 9.0 by adding an acetic acid solution, and finally separating the enzyme produced in crystal form.
 7. A process for the production of a new alkaline protease, according to claim 2, in which a purificatioN comprises the steps of precipitating the filtrate of the culture broth by adding acetone, dissolving the precipitate in the minimum required amount of distilled water, fractionally precipitating with ammonium sulfate, collecting the precipitate by centrifugation or filtration, dissolving said precipitate in the minimum required amount of distilled water at pH 9.0, readjusting the solution to between pH 8.2 and 8.5 with Paliztsch borate buffer (pH 8.2) or acetic acid solution, and finally separating the enzyme produced in crystal form.
 8. A process for the production of a new alkaline protease, according to claim 2, which comprises steps of adjusting the pH of the filtrate of the culture broth to 8.2 with Paliztsch borate buffer or acetic acid solution, and separating therefrom the enzyme in crystal forms.
 9. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of fractionally precipitating the filtrate of the culture broth with ammonium sulfate, collecting the precipitate by centrifugation or filtration, adjusting the pH to 9.0, dissolving it in the minimum required amount of distilled water, and purifying on a Sephadex column.
 10. A process for the production of a new alkaline protease, according to claim 2, in which a purification comprises the steps of precipitation the filtrate of the culture broth with a solvent selected from the group consisting of acetone, methanol and ethanol, adjusting the precipitate to pH 9.0, dissolving it in distilled water, again precipitating it with the above solvent, and separating the pure enzyme crystals. 